This project applies genomic and high-throughput genetic strategies to the global health emergency of tuberculosis. It creates new resources that will bridge clinic and laboratory, and quicken research for new, cheaper, and more effective treatments and vaccines. Tuberculosis (TB) is the world's most deadly bacterial infection and kills two million persons each year. It is estimated that between one-half and one-third of the world's population is latently infected with M. tb. In the US there are currently between 10,000 and 20,000 active TB cases per year. Treatment of even completely drug-susceptible strains currently requires 6 months of multiple antibiotics taken daily and often involves deleterious side effects. Multiply-drug-resistant or MDR) and extensively drug resistant XDR strains that resist some, most, or even all current chemotherapies are increasingly common. There is a deadly interaction of TB with AIDS, which makes the effect of both epidemics more harmful. Thus, the need for new and more effective chemotherapies and vaccines against TB is urgent. The research tools available for M. tb are not commensurate with the scale of the problem or what is available in "model" microorganisms such as E. coli and yeast. This project will create a new genetic infrastructure in service of the global fight against tuberculosis. The core of the project is to combine genetic engineering and newly refined genetic methods in M. tb to make three large sets each of 4,332 M. tb strains, with each strain deleted in a defined way for a single gene. Furthermore, each deletion is marked by a unique barcode sequence allowing quantification of the proportion of each genotype in a mixed population. Bar coded deletion libraries will be built in two different genetic backgrounds: 1) H37Rv, however, is the standard laboratory strain;2) mc26320, a nonpathogenic derivative of H37Rv. H37Rv is a pathogen and it must be worked with under BSL3 conditions, whereas mc26320 requires less stringent BSL2 containment and is an important base strain for vaccine cell studies. These strain sets and related resources will be important tools for M. tb genetics. The genetic resources created by this project will be made available to all qualified researchers worldwide via an NIH-designated depository and distribution center. It is already known that certain M. tb genes are key to questions of antibiotic tolerance, biofilm formation, interaction of M. tb with the host immune system, and pathogenic growth. Application of the library will finally allow an exhaustive enumeration of all M. tb genes that are relevant for these key aspects of tuberculosis and its treatment. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis, the causative agent of Tuberculosis (TB) is responsible for a worldwide health burden. Genetic tools are needed to better understand the tubercle bacilli so better therapeutics including drugs and vaccines can be developed. We propose to create a set of single gene knockout for every gene of Mycobacterium tuberculosis in order to better understand drug resistance and pathogenesis.